Polynucleotide vaccines are an innovative approach for inducing protective immunity against specific diseases, both generating neutralizing antibodies as well as activating the more preferable cell-mediated immune responses (Montgomery, D. L. et al., 1993, Cell Biol. 169:244-247; Ulmer, J. B. et al., 1993, Science 259:1745-1749). Plasmid DNA comprising a gene encoding an antigen of interest and a promoter active in mammalian cells is administered to the body and internalized by muscle cells. The antigen DNA is transcribed and translated, and the expressed protein is transported to the cell surface for T-cell presentation. Preclinical immunogenicity and efficacy of DNA vaccines in disease models have been demonstrated for a number of infectious diseases (for a review, see Gurunathan, S. et al., 2000, Ann. Rev. Immunol. 18:927-974). Plasmid DNA has additionally been approved for gene therapy treatment, encompassing the administration of a functional gene into the body, delivery of said gene to a target cell, and expression of the therapeutic product with the intent to selectively modulate disease conditions. Thus, gene therapy represents an alternative for the prevention, treatment or cure of genetic defects. Many plasmid DNA-based gene therapy clinical trials have been initiated (for a review, see Mountain, A., 2000, TIBTECH 18:119-128; and Ferber, D., 2001, Science 294:1638-1642).
The manufacture and purification of large quantities of pharmaceutical-grade plasmid DNA is crucial to the applicability of both polynucleotide vaccine and gene therapy protocols. The potential number of human users for DNA vaccines or gene therapy treatments to combat disease, as part of either a prophylactic or therapeutic regimen is very large, creating a high demand for clinical-grade plasmid DNA. Thus, high yield plasmid DNA production and purification processes are necessary to fully develop and exploit the advantages that both DNA vaccine and gene therapy treatment options have to offer (Shamlou, P. A., 2003, Biotechnol. Appl. Biochem. 77: 207-218). Despite prior investigations into small-scale plasmid DNA purification methodologies, scaling-up the manufacture and purification of clinical-grade plasmid DNA has been proven to be problematic (Prazeres, D. M. F. et al., 1999, TIBTECH 17:169-174). In addition, innovative large-scale manufacturing processes must balance optimization and economic concerns against demand and need for speed-to-market (Shamlou, 2003, supra). The present invention discloses a highly productive, scalable and reproducible process for the purification of plasmid DNA that reduces production costs and increases process robustness. The process discloses a new lysis and lysate clarification procedure which includes polymer flocculation of host cell debris. This new lysis and flocculation procedure may be combined with a novel, downstream polishing step encompassing precipitation of plasmid DNA (utilizing polyethylene glycol or alcohols) and subsequent microfiltration under a tangential flow filtration mode.
Chemical flocculation is commonly used to isolate bacterial cells from growth medium, representing a less expensive alternative to centrifugation (see, e.g., Lee, J. and C. V. Viswanathan, 1974, Lab. Pract. 23:297-298; Cumming, R. H., et al., 1996, Bioseparation 6:17-23). The mechanism of flocculation is complex, depending on many variables such as temperature, ionic environment, physiological age, flocculant, surface shear and material to be flocculated (McGregor, W. C. and R. K. Finn, 1969, Biotechnol. Bioeng. 11: 127-138). Only a few studies have analyzed this mechanism in relation to bacterial cell debris. Persson, I.-L. and B. Lindman (“Flocculation of Cell Debris for Improved Separation by Centrifugation,” Flocculation in Biotechnology and Separation Systems, Ed. Y. A. Attia, Amsterdam: Elsevier, 1987, 429-439) used a combination of cationic polyelectrolytes, chitosan and polyethylene imine, to flocculate E. coli cell debris in laboratory and pilot plant studies. Positively charged polymeric particles have also been used to flocculate E. coli cell debris (Kim, C. W., et al., “Removal of Cell and Cell Debris by Electrostatic Adsorption of Positively Charged Polymeric Particles,” Flocculation in Biotechnology and Separation Systems, Ed. Y. A. Attia, Amsterdam: Elsevier, 1987, 429-439). However, the ability of using a polymer flocculant to generate a clarified bacterial lysate for scalable process design has not been recognized.
The last step in purifying clinical grade plasmid DNA from microbial cell fermentation encompasses removing any residual, host cell-derived impurities and/or process contaminants carried over from prior upstream purification steps, as well as concentrating the final product and buffer exchange. The present invention utilizes well known methods of precipitating DNA with polyethylene glycol (see, e.g., L is, J. T. and R. Schleif, 1975, Nucleic Acids Res. 2:383-389; Sadhu, C. and L. Gedamu, 1988, Biotechniques 6:20-21; Yeung, M. C. and A. S. Lau, 1993, Biotechniques 15:381-382; and Horn, N. A. et al., 1995, Hum. Gene Ther. 6:565-573) or alcohols (see, e.g., Wallace, D. M., “Precipitation of Nucleic Acids,” Methods in Enzymology: Guide to Molecular Cloning Techniques, Eds. S. L. Berger and A. R. Kimmel, 1987, 41-48; Serghini, M. A. et al., 1989, Nucleic Acids Res. 17:3604) in combination with microfiltration in a tangential flow filtration mode in a final polishing procedure to complete plasmid DNA purification. This process eliminates the high recirculation rates and large membrane areas required for ultrafiltration procedures commonly used in final polishing processes.
U.S. Pat. No. 5,561,064, issued to Marquet, M. et al. on Oct. 1, 1996, discloses a differential polyethylene glycol (“PEG”) precipitation strategy utilized during the purification of pharmaceutical-grade plasmid DNA. Importantly, the first PEG precipitation step occurs after the production of a clarified lysate and prior to size exclusion of anion exchange chromatography.
U.S. Pat. No. 5,707,812, issued to Horn, N. et al. on Jan. 13, 1998, discloses the use of PEG as a condensation agent to enhance the binding of plasmid DNA to a chromatographic matrix, from which said DNA is subsequently eluted in a salt buffer containing PEG.